To know how many proteins assemble together at the nanoscale is fundamental for understanding protein function. Sometimes, proteins must be in an "oligomeric" state to be functional, although "oligomerization" of certain proteins can also lead to diseases. The ability to determine protein stoichiometry and monitor changes in the balance between monomeric, dimeric and multi-meric proteins can allow scientists to see the differences between a properly functioning cell and a diseased cell. Therefore, there is a great interest in being able to count proteins and determine their stoichiometry.
In a recent study carried out at ICFO, the Institute of Photonic Sciences, the research group of Advanced fluorescence imaging and biophysics, led by Nest Fellow Dr. Melike Lakadamyali was able to quantify the photoactivation efficiency of all the known "ir-reversibly photoswitching fluorescent proteins" and establish a proper detailed reference framework for determining protein stoichiometry. To do this, they used a nanotemplate of known stoichiometry (the human Glycine receptor expressed in Xenopus oocytes) and studied several fluorescent proteins to see the percentage of proteins that was photoactivated. The results of this study have recently been published in Nature Methods.
"Molecular counting" is becoming a closer reality thanks to the discovery of photoactivatable fluorescent proteins and the development of super resolution microscopy. Photoactivatable flourescent proteins change their fluorescence property from dark to bright when exposed to light (i.e. laser light). Through the use of localization-based super-resolution microscopy, researchers are able to photoactivate, image and follow these genetically encoded fluorescent proteins, one at a time, to study what is happening inside a cell at the molecular level. However, despite the "molecular counting" ability that seems intrinsic to the imaging strategy (activating one fluorescent protein at a time should also allow counting how many total fluorescent proteins exist) relating the number of counted fluorescent proteins to actual protein stoichiometry has been difficult. One important reason for this difficult task in counting has been the fact that, until recently, it was not known whether all fluorescent proteins become bright when exposed to laser light. Failure to photoactivate would lead to undercounting since a fraction of probes would be dark and never appear in the image.